BIOSYNTHESIS OF ADENOVIRUS EARLY RNAS

癌细胞的异常特性部分是由于 某些转录因子(TF)的不适当激活和 使其他人失去活力。了解TFS的功能应该允许 治疗方法的设计可以改变导致癌症的异常的TF 致癌作用。大多数调节因子是具有不同DNA的模块化蛋白 结合域和激活域。DNA结合的机制 域的功能已经被很好地理解了，但关于它是如何实现的却知之甚少 激活域起作用。激活域刺激PolIl 由一般组成的复杂的预起爆复合体引发 TFS和PolIl.我们建议研究该基因的激活区域。 强病毒激活剂2腺病毒E1a、Epstein-Barr病毒Zta和 单纯疱疹病毒VP16以及重要的肿瘤抑制因子 P53。这些研究取决于提纯功能性TFIID的能力，即 由TATA结合组成的复杂通用转录因子 启动预引发复合体组装的蛋白质(TBP)和TAFs 拿着塔塔盒对着推销员。一种次要的核蛋白CR3BP已经被 与E1a辅活化子的预测性质相一致：它 结合wt E1a激活域，但不结合有缺陷的突变体 在激活时确实结合了TBP。如果额外的实验是 与E1a辅活化子功能一致，编码CR3BP的cDNA将 被克隆并用于分析其转录活性。上的区域 与E1a和其他激活结构域相互作用的TBP表面， 将通过引入氨基酸来分析一般的TFS和TAFs 它的91个表面氨基酸残基中的每一个都进行了替换 而不是联系DNA。TFIID包含突变体Tbps，可结合普通TF和 正常但有缺陷的TAFs将被激活转录 分离并用于激活结构域结合和分析 预引发复合体组件以确定哪一步被激活 组装、POLII启动或启动子清除有缺陷。Zta 激活TFIID和TFIIA在启动子DNA上的组装，但这 刺激不足以完全解释Zta的激活。 在D-A组装之后的预引发复合体组装中的步骤将 用能够分解DNA蛋白质的琼脂糖凝胶进行检测 ~gt；10(6)Da络合物及其凝胶过滤检测因子 与质粒模板结合。启动POL II和 还将分析启动子清除情况。类似的研究将分析 E1a和P53的激活和激活剂的组合激活 关于具有两种类型激活剂结合位点的合成模板。 针对最近克隆的POL亚单位产生的特异性抗体 将使用III因子TFIIIC来分析TFIIIC的机制 应对病毒感染和生长因子的监管。

The abnormal properties of cancer cells are due in part to the inappropriate activation of some transcription factors (TFs) and the inactivation of others. Understanding how TFs function should allow the design of therapies that modify the abnormal TFs that contribute to oncogenesis. Most regulatory TFs are modular proteins with distinct DNA binding and activation domains. The mechanisms by which DNA binding domains function are well understood, but little is known about how activation domains function. Activation domains stimulate pol Il initiation from a complicated preinitiation complex composed of general TFs and pol Il. We propose to study the activation domains of the strong viral activators adenovirus 2 E1A, Epstein-Barr Virus Zta, and herpes simplex virus VP16; as well as the important tumor suppressor p53. The studies depend on the ability to purify functional TFIID, the complex general transcription factor composed of the TATA-binding protein (TBP) and TAFs that initiates preinitiation complex assembly at promoters with a TATA-box. A minor nuclear protein, CR3BP, has been identified with the predicted properties of an E1A coactivator: it binds the wt E1A activation domain, but not to point mutants defective in activation that do bind TBP. If additional experimentation is consistent with E1A coactivator function, a cDNA encoding CR3BP will be cloned and used to analyze its transcriptional activity. Regions on the surface of TBP that interact with E1A and other activation domains, general TFs and TAFs will be analyzed by introducing amino acid substitutions into each of its 91 surface amino acid residues that do not contact DNA. TFIID containing mutant TBPs that bind general TFs and TAFs normally but are defective for activated transcription will be isolated and used in assays of activation domain binding and preinitiation complex assembly to determine which step in activated assembly, pol II initiation or promoter clearance is defective. Zta activates assembly of TFIID and TFIIA on promoter DNA, but this stimulation is not sufficient to account completely for Zta activation. Steps in preinitiation complex assembly subsequent to D-A assembly will be assayed using agarose gels capable of resolving DNA protein complexes of >10(6) Da and a gel filtration assay to detect factor binding to plasmid templates. Activation of pol II initiation and promoter clearance will also be analyzed. Similar studies will analyze activation by E1A and p53 and activation by combinations of activators on synthetic templates with binding sites for two types of activators. Specific antibodies raised against a recently cloned subunit of the pol III factor TFIIIC will be used to analyze the mechanism of TFIIIC regulation in response to viral infection and growth factors.